Effective therapies for CRC still remain a critical unmet medical need. One of the most important drivers of CRC is K-Ras, which is mutated in 40% of human CRCs. Indeed, Ras proteins play essential roles as molecular switches, controlling cell proliferation, growth, differentiation, and apoptosis. Deregulation of the Ras signaling pathway by activating mutations, overexpression, or upstream activation is common in many human tumors. Thus, K-Ras represents a highly validated and important target for CRC and a wide variety of other cancers. Although a number of different approaches have been attempted to target upstream or downstream proteins in the K-Ras signaling pathway, it would be ideal to target K-Ras itself However, KRas is considered to be a poor drug target. In order to test whether K-Ras could be druggable with a small molecule, we cloned, expressed, isotopically labeled, and purified K-Ras (G12D) and conducted a fragment-based screen on GDP- and GTP-bound K-Ras using NMR. In these screens we identified over 100 small molecules that bind to K-Ras, suggesting that K-Ras may be a druggable target. In addition, we obtained several X-ray crystal structures of K-Ras bound to the hits identified in the screen, which we are currently using to guide the synthesis of K-Ras inhibitors. We propose to discover small molecules that potently bind to K-Ras, inhibit its functions, and are highly efficacious against in vivo CRC tumor models with the goal of discovering a compound that is suitable for entry into a CRC clinical trial. We hypothesize that a K-Ras inhibitor will be highly effective for treating CRC patients. Aim 1. Generate lead compounds that bind K-RAS tightly (nM) from our fragment-based screens and recently determined three-dimensional structures of K-RAS/inhibitor complexes using iterative structure-based design. Aim 2. Optimize lead K-RAS binders for their K-RAS inhibitory effects in biochemical assays and their cell-based activities against colon cancer cells. Aim 3. Prioritize K-RAS inhibitors with excellent pharmaceutical properties that are efficacious in vivo using a new stem cell-derived, tamoxifen-inducible Cre driver (Lrig1-CreERT2) mouse to activate mutant KRAS in the mouse colon. Select a compound that is suitable for a clinical trial in colon cancer by the end of the granting period.